This A2 application has the long-term goals of defining the mechanisms that regulate the formation of the major reciprocal circuit in the forebrain. The present proposal represents a new project in which the development of cortico-thalamic (CT) connectivity will be studied. The CT and thalamo-cortical (TC) circuits are precisely organized topographically between and within target cortical areas and nuclei. There are virtually no studies examining axon guidance mechanisms of CT circuitry, even though they are ~10 times more numerous than TC projections and comprise well over half of the synaptic input onto thalamic relay neurons. Topographic reciprocity of CT connections is essential for spatial tuning of sensory processing and integration, both of which are disrupted in a variety of neurodevelopmental disorders, including autism, schizophrenia and attention deficit hyperactivity disorder. In a series of new studies, we used in utero electroporation-mediated gene transfer to label selectively CT axonal populations and, at the same time, disrupt putative guidance cues in the embryonic cerebral cortex of the mouse. The altered expression of Eph receptor A7 (EphA7) by overexpression or siRNA-mediated reduction disrupts within-nucleus CT targeting, but not the organization of between-nucleus projections. Moreover, cortical EphA disruption does not produce topographic changes in TC projections, which will allow us to create circuits that have unmatched reciprocal topography. We hypothesize that the development of efferent cortical axons that project to reciprocal nuclei of the dorsal thalamus (DT) utilize mechanisms distinct from TC projections for two components of circuit formation: 1) Slit-Robo cues to reach their appropriate thalamic target nucleus and 2) Eph-ephrin cues to establish dorsoventral (D-V) and mediolateral (M-L) topography within the appropriate target nucleus. Three aims are proposed to examine the development of CT circuitry. In the first aim, the relative contributions of EphA7 and EphAs4,1 and 2 to within-nucleus mapping along D-V and M-L axes will be examined using in utero electroporation methods. In the second aim, two families of EphA cytoplasmic effectors, the ephexins and Vavs, which have a role in retino-thalamic patterning, will be studied to determine the role of each family of guanine nucleotide exchange factors in mediating TC and CT circuit development. Ephexin mutant mice will be analyzed for alterations in CT and TC organization, in a collaborative project with Dr. M. Greenberg. In the third aim, the role of Robol and 2 activation on cortical neurons, by DT slits, in mediating between-nucleus topography will be tested using in utero electroporation strategies and by examining slit1;slit2 null mice. The proposed studies have broad relevance for public health because they will lead to a more detailed understanding of the development of thalamic-cortical loops, a likely substrate for altered information processing that accompanies disorders of neurodevelopment, psychiatry and drug abuse.